PROJECT SUMMARY/ABSTRACT Fragile X Syndrome is the most common heritable form of intellectual disability and leading monogenetic cause of autism. It has been estimated that 1 in 5,000 males are born with Fragile X Syndrome. This disease is caused by loss of the Fragile X Mental Retardation Protein (FMRP), a repressor of mRNA translation into protein products. In addition to protein synthesis, FMRP has been observed to affect dendritic spine development. Few studies have demonstrated how the repressive activity of FMRP itself is regulated. FMRP is ubiquitinated in response to glutamate receptor stimulation, providing a potential mechanism to dynamically remove translational repression. The researchers propose that ubiquitination may be a regulatory mechanism to reverse FMRP's repression of protein synthesis. The effects of FMRP ubiquitination on global protein synthesis and downstream pathways are unknown. Recently, the E3 ligase anaphase-promoting complex (APC) and its regulatory subunit Cdh1 were shown to ubiquitinate FMRP. Preliminary evidence suggests that ubiquitination of FMRP contributes to the disassociation of the RNA-induced silencing complex (RISC) from target mRNA and subsequent protein synthesis. These data suggest that activity of FMRP and its role as a repressor of translation may be modulated by ubiquitination. Given that FMRP regulates the synthesis of a large and diverse set of proteins that are critical for signaling and synaptic development, ubiquitination of FMRP may drive differential dendritic spine morphology. The central hypothesis is that Cdh1-APC mediated ubiquitination of FMRP regulates protein synthesis and dendritic spine morphology through the disassociation of RISC from targeted mRNA. Aim 1 will test the hypothesis that ubiquitination of FMRP leads to increased protein synthesis and more immature spines. Aim 2 will investigate the role of a specific E3 ligase, Cdh1-APC, in downstream FMRP-dependent effects, including protein synthesis and dendritic morphology. Ubiquitination- resistant mutants and Cdh1 shRNA will be utilized to study these mechanisms. The trainee will master a wide range of molecular and cellular biology techniques including cloning, AHA pulse labeling, proximity-ligation assays, immunofluorescence, and automated spine morphology analysis. The proposed research will provide insight into the currently unknown regulation of mRNAs encoding synaptic proteins targeted by FMRP, and will elucidate novel mechanisms underlying translational regulation. This research will provide a foundation for future development of disease mechanism-based therapeutic strategies for FXS and other neurodevelopmental disorders.